see manuscript in preparation Dressaire et al. 2008. Growth rate regulated genes and their wide involvement in the Lactococcus lactis stress responses
Extracted molecule
total RNA
Extraction protocol
A volume of culture corresponding to 6 mg (dry weight) of cells was frozen immediately in liquid nitrogen. Before cell lysis, each sample was centrifuged (4°C, 5 min, 8000 rpm), washed with 1 ml TE buffer (Tris-HCl 10 mM pH 8, EDTA 1 mM) and resuspended in 500 µl TE buffer. Cells were disrupted at 4 °C (3 cycles of 1 min interspaced by 3 min cooling periods) on a minibead beater (Biospec Products) with glass beads (0.6 g), 50 µl SDS (10 %), and 500 µl phenol (pH 4.7). After centrifugation to eliminate cell debris and phenol (4°C, 25 min, 13000 rpm), half of the aqueous phase containing RNA was extracted with RNeasy midi kit (Qiagen), including the DNase I treatment described in the manufacturer’s instructions. RNA was quantified at 260 and 280 nm and RNA quality was controlled on electrophoresis agarose gel in denaturing conditions.
Label
33P
Label protocol
For cDNA synthesis, 10 or 15 µg of total RNA was mixed with 1 µl of random hexamer primers (Sigma Genosys, 500 ng.µl-1), 1 µl of Eurogentec L. lactis open reading frame specific primers (500 ng.µl-1) and sterile water to a final volume of 24 µl. The mixture was heated at 70°C for 5 min and then immediately cooled to 4°C. Reverse transcription was performed for 1 h at 42°C in a total volume of 50 µl, with SuperScriptII reverse transcriptase (300 U, Life Technologies), dithiotreitol (10 mM, Life Technologies), dATP, dGTP, dTTP (0.3 mM each), and [-33P]dCTP (50 µCi, Amersham Biosciences) and 5X first strand buffer (1X, Life Technologies). After 1 h, SuperScriptII reverse transcriptase (100 U) and dCTP (0.1 mM) were added and the reaction performed for one more hour at 42°C. Reaction was stopped by heating (15 min at 70°C), and the remaining RNA was hydrolyzed by RNaseH (2 U, 20 min, 37°C, Life Technologies). Before hybridization, labeled cDNA was purified on Microspin G25 columns (Amersham Biosciences) according to the manufacturer’s instructions.
Hybridization protocol
L. lactis IL1403 specific PCR products were spotted in duplicate on positively charged nylon membranes (4 deposits per spot, Plateforme Génomique, Toulouse). A single PCR per ORF was designed by Eurogentec, PCRs covered about 71 % of each ORF length (mean length 535 bp and final concentration between 40 and 180 µg.ml-1). 2053 ORFs of the 2310 identified on the genome were effectively available. Prior to hybridization, membranes were washed for 5 min in 50 ml of 2X SSPE (SSPE 1X is 0.18 mM NaCl, 1 mM EDTA, and 10 mM phosphate buffer pH 7.2) and prehybridized for 3 h at 68°C in 5 ml of hybridization buffer (5X SSPE, 2 % SDS, 1X Denhardt’s reagent). Labeled cDNA was heated for 10 min at 95°C and immediately cooled at 4°C for 5 min. Membrane hybridization was carried out for 15 h at 68°C with 5 ml of hybridization buffer containing labeled and denatured cDNA. Membranes were then washed 3 times in 50 ml of washing solution (0.5X SSPE, 0.2 % SDS) for 5 min at room temperature and 3 times in 50 ml of preheated washing solution for 20 min at 68°C. Dried membranes were exposed to a phosphoimager screen for 3 days and scanned with a phosphofluoroimager (Storm 860, Molecular Dynamics).
Scan protocol
Hybridization signals were quantified, assigned to gene names, and statistically treated with the Bioplot software (developed by S. Sokol in Plateforme Génomique, Toulouse, http://biopuce.insa-toulouse.fr/). Local background was removed and signals were normalized by the mean intensity of the membrane.
Description
see Redon et al. 2005. Role of mRNA stability during genome-wide adaptation of Lactococcus lactis to carbon starvation. J Biol Chem 280(43):36380-385.
Data processing
Local background was removed from the raw signals with the Bioplot software (Biochips Platform, INSA Toulouse, France, http://biopuce.insa-toulouse.fr).. Data were normalized by the all spot’s mean with Bioplot software.
